Dual-labeled Probes for qPCR



 

Overview
>>to the top

Microsynth's offers a wide selection of fluorophores and quenchers which will support most real-time quantitative PCR instruments and multiplex analysis systems. Here are some arguments to use dual-labeled probes, especially those from Microsynth.


Main Product Features and Benefits:

  • Broad portfolio of probes: availability of various fluorophore and quencher combinations (including internal quencher)
  • High quality: all probes are MALDI-TOF controlled before shipment
  • Minimum delivery guarantee (delivered amount is on average at least 3 times higher)
  • Fast delivery (within 3 working days on average)
  • Competitive and transparent pricing: prices for dual-labeled probes include all DNA bases, fluorophores & quenchers as well as a thorough purification procedure (HPLC or even PAGE)
  • Free of charge: free probe design service available; design of dual-labeled probes and primer pairs on request
  • Additional services:
    • Design, synthesis and functionality testing of primer-probe sets for real-time PCR (see also the flyer "Customized qPCR Assays" under Related Downloads)
    • C-5 propynyl modification of your probe (see also the flyer "MGB and LNA Substitutes" under Related Downloads)¹
    • Aliquotation of probes and shipment within 5 tubes on request2

Important – Please Note!

If your qPCR experiment requires the use of FRET hybridization probes (probes for LightCycler®) or Molecular Beacons, Microsynth can of course help you with respect to design and synthesis of high-quality primer-probe sets. Interested to obtain more information and/or to receive an offer? Then please do not hesitate to contact us.



1 Nucleic acids with enhanced hybridization properties (e.g. C5-propynes, MGBs, LNAs) are usually used to:

  • Increase the thermal stability and hybridization specificity of your probe
  • Get greater accuracy in gene quantitation and allelic discrimination
  • Facilitate the probe design of problematic target sequences

2 If you wish your probe within 5 separate tubes (only valid for 0.2 and 1.0 µmol synthesis scales) please proceed as follows during the ordering process: Either immediately return the order confirmation e-mail and note “please ship dual-labeled probes in 5 aliquots”; or alternatively write “please ship dual-labeled probes in 5 aliquots” into the field “special comments” when using the upload entry mode.



 

Fluorophores and Quenchers
>>to the top

Dual-labeled probes (also called hydrolyzation probes or TaqMan® probes) are usually labeled with the reporter at the end 5´ and with the quencher placed at the end 3´. Choice of the reporter depends on excitation and emission channels of the used real-time PCR cycler (almost all real-time PCR cyclers provide a channel for fluorescein - FAM).

Today, BHQ (Black Hole Quencher) type quenchers are usually used as quenchers; they quench a broad spectrum of fluorophores and show no autofluorescence. For more detailed information regarding the use and interplay of the most common fluorophores/ reporters and quenchers, please download the flyer “Fluorophores and Quenchers”.

To learn more about our prices, please login to our webshop and download the current price list. Microsynth supplies hydrolyzation probes (single- or double-quenched) which are specified in more detail in the tables below.

Product Characteristics:
Double-quenched probes:
In traditional single-quenched probes, dye and quencher are separated by 20-30 nucleotides. Our internal quencher IQ-500 shortens the distance between dye and quencher, and in concert with the 3' quencher, provides a higher degree of quenching, a lower background as well as an increase in signal detection. When having a 20-30 bp distance between 5' dye and 3' quencher, we recommend to use a 9 bp distance from the dye. In case you need help in the design of your qPCR assay, let us know.   

5‘ Label

Internal & 3‘ Label

0.04 µmol scale1 0.2 µmol scale1

1.0 µmol scale1

Minimum yield2 Average yield2

Minimum yield2

Average yield2

Minimum yield2

Average yield2

FAM

IQ-500 with BHQ-1 or TAMRA

n/a n/a

2 OD

10 nmol

8 OD

40 nmol

8 OD

40 nmol

25 OD

125 nmol


Single-quenched probes: 

5‘ Label

3‘ Label

0.04 µmol scale1 0.2 µmol scale1

1.0 µmol scale1

Minimum yield2 Average yield2

Minimum yield2

Average yield2

Minimum yield2

Average yield2

FAM

TAMRA

1 OD

5 nmol

3 OD

15 nmol

2 OD

10 nmol

8 OD

40 nmol

10 OD

50 nmol

30 OD

150 nmol

BHQ-1

9 OD

45 nmol

8 OD

40 nmol

25 OD

125 nmol

HEX

TET

TAMRA n/a n/a

2 OD

10 nmol

8 OD

40 nmol

10 OD

50 nmol

30 OD

150 nmol

AlexaFluor 350

FAM

HEX

TET

Dabcyl

n/a n/a

2 OD

10 nmol

5 OD

25 nmol

10 OD

50 nmol

20 OD

100 nmol

AlexaFluor 350

HEX

JOE

TET

ATTO 425/532

Yakima Yellow

(equivalent to VIC)

BHQ-1

n/a n/a

1.5 OD

7.5 nmol

6 OD

30 nmol

8 OD

40 nmol

25 OD

125 nmol

ATTO 647N

ATTO 620

ATTO 550

(equivalent to NED)

Cy3/Cy5

Dyomics 681

ROX

TAMRA

Texas Red

BHQ-2

n/a n/a

1.5 OD

7.5 nmol

4 OD

20 nmol

8 OD

40 nmol

15 OD

75 nmol

1 The synthesis scale represents the initial amount of 3' bases (starting material).

2 Yields indicated in OD260 apply to a probe consisting of 20 DNA bases; Calculation: 1 OD = 5 nmol (please note that this calculation is based on sequences with virtually homogenous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.)


If your desired fluorophore or quencher is not listed in the tables above, please contact us for availability and pricing.

Potential Applications:

• Gene expression
• Multiplex assay development
• Pathogen detection
• Viral load quantitation
• Microarray validation
• si/shRNA knockdown
• Gene copy determination
• Mutation detection
• Allelic discrimination
• SNP detection
• Human and veterinary diagnostics
• GMO analysis


Some Remarks to the Correlation of Yield and Number of Reactions


In the case of real-time PCR, usually one measurement or reaction requires 20 µl of a solution containing the probe at a concentration of 0.2 pmol/µl. As a consequence, one reaction consumes 4 pmol of a probe. For instance, if you order a 5’ FAM + 3’ BHQ-1 probe at 0.2 µmol synthesis scale we deliver at least 10 nmol of the specified probe to you. Therefore, you can theoretically perform at least 2’500 reactions (whereas the average delivered amount of ~45 nmol is sufficient for ~10'000 reactions).

Calculation of the number of reactions for real-time PCR (quantity of probe: 4 pmol):

# reactions = delivered amount [nmol] * 1’000 / 4


How to Order?

  • Enter our webshop
  • Click on DNA in the "Oligonucleotide" domain
  • Select either Normal Entry in order to type or copy/paste the desired sequence information etc. or alternatively select Upload Entry by using our convenient Excel Template (can be downloaded during ordering)
  • Single-quenched probes: Select your desired 5', 3' modification as well as purification, and your oligo is recognized and processed as a single-quenched probe.
    Double-quenched probes: Include ordering symbol "5" into your sequence (e.g. catattgaa5actgggttaacggaatt) and choose "Internal Quencher 500" under Inner Modification ("5=…"). Select your desired 5', 3' modification as well as purification, and your oligo is recognized and processed as a double-quenched probe.


 

Design Service for Primer Probes Sets – Free of Charge
>>to the top

Rely on our vast experience in the area of oligonucleotide synthesis and real-time PCR (allele-specific and exon-exon junction overlapping real-time PCR, multiplex kit development, SNP detection) and and let us design and produce your primer probe sets.


How to Proceed:

  • Contact us by e-mail and
  • Provide us with the accession number or alternatively with the gene sequence of your target gene
  • Do not forget to mention the desired synthesis scale as well as 5’- and 3’-labels

Conditions:

  • Design service is free of charge for up to 5 primer probe sets per day and customer

If you want to design your primer probes sets on your own, you will find some useful information under Design of Primer Probe Sets in the submenu “Hints and Tips”.






rechte sp
Questions?
Write an e-mail or call us at +41-71-722 83 33

rechte sp
Related Links

Related Downloads
Customized qPCR Assays.pdf
Informs how Microsynth designs, synthesizes and tests the functionality of primer-probe sets for real-time PCR

DoubleQuenchedProbes.pdf
Shows the principle and advantage of using double-quenched qPCR probes

MGB_LNA_Substitutes.pdf
Informs about alternatives for MGA_LNA probes

Notification C-5 Propynes.pdf
Notification about the usage of C-5 Propynes for research only.


to the top